Quinonoid dihydrobiopterin, an important metabolic intermediate of biopterin cofactor in the aromatic hydroxylation of amino acids

Tetrahedron Letters, Vo1.27, Printed in Great Britain

Quinonoid

No.5,

an Important

Dihydrobiopterin, Cofactor

Sadao Department

in the Aromatic

Matsuura*,

of Chemistry

Shizuaki

College

of

p-Quinonoid

from

dihydrochloride potassium

In

the

Sugimoto

General

Education,

Nagoya

Nagoya

Several

were

of

Acids

and Takashi

University

464, Japan

hydrochloride

by hydrogenperoxide

biosynthesis

of Amino

of Biopterin

was

(65)-5,6,7,8_tetrahtdrobiopterin

iodide.

dihydrobiopterin

Intermediate

Murata,

(6+)-dihydrobiopterin

synthesized

0040-4039/86 $3.00 + .OO 01986 Pergamon Press Ltd.

Metabolic

Hydroxylation

Chikusa-ku,

Abstract:

1986

pp 585-588,

in the

characters

presence

of

of

quinonoid

examined.

neurotransmitting

catecholamines,

the

hydroxyla-

tion of phenylalanine

and tyrosine requires (6R)-5,6,7,8_tetrahydrobiopterin cofactor. 1 During the enzymatic reactions, 1 is oxidized

(1) as the natural to quinonoid pterin

2 will

pathway,

and

(3)

the existence

enzymatic because

its

spectroscopic

respective

would

and

have

(0.033 g, 0.20 mmol) was

added

ethanol.

30% H202 was By

of

(0.2 ml,

collected

this

0.354 g (64% yield)

washing

active

deduced

from

are

of

2 has

unstable

synthesis

2.0 mmol) by

at 0 'C.

iodine

was

dihydrobiopterin

585

washed

unsuccessful

completely

and

and

6,7-

products

of we

of 2.

2.0 mmol) (5 ml),

stirring with

of the

communication

properties

After

and

study

6-methyl-

water

7,8la

biological

(0.630 g,

ice chilled

filtration

procedure,

of quinonoid

into

stable

oxidation

In this

and some

recycling

reductase.

been

1. Thence

on quinonoid

(1) dihydrochloride4

the

the kinetic

of

the

such more

towards

preparation

performed

dissolved

to 1 by dihydro-

of

to thermodynamically

more

which

back

absence

pseudocofactors.2'3

a chemical

were

is reduced

In the

unavailability

been

both

tetrahydropterin

like to describe

no

reaction,

instability studies

isomerize is

of 2 has been

(6lj)-Tetrahydrobiopterin

precipitate

(or NADH).la

which

hydroxylation of

dimethyldihydropterins, the

NADPH

spontaneously

dihydrobiopterin Although

(2), which

(6R)-dihydrobiopterin

reductase

and

for 5 min, cold

water

removed,

(2) hydrochloride

KI

to which the and

leaving as ivory

586

H202-KI W NADPH with or without

1

P” 7.0

enzyme

_,,ty---ficH3 ‘N

II

3 needles. 5

The

at 1740 cm-l The

IR spectrum

for the quinonoid

obtained

probably adducts

or

weak

because

the

with

standing

2

could

a typical

band

structure.3 be

analyses

without NaHS03, min. 7 The main

had

in any

aqueous

stored

when

compound

NaHS03

compound

were

without

significant

cation

to 2 was observed

proceeded

during

may

decomposed

decomposition

products

some

a pH

exchange

HPLC

semi

by oxidation

to decompose.

using

Thus,

except This

is

stable after all

the

3.2 buffer containing 6 column. A single and

on chromatograms

spontaneously were

examined NaHS03.

form

the chromatography.

the compound

On

of solutions containing

is destroyed

2 starts

performed

on a strong

kind

solution

dihydro

In fact,

corresponding

decompositions

rapidly

quinonoid

the dihydro

as the eluant

sharp peak

very acidic

NaHS03.

in air,

chromatographic NaHS03

of

exhibited

'Cformorethantwomonthsinatightlysealedbottle.

2 decomposed

in a neutral

dihydropterin

crystals

decompositionat contrary,

(KBr disk) of the compound

unexpectedly

indicating

that no

In a pH 3.5 solution with

a half

life

3.5

(6R)-tetrahydrobio-

587 pterin

(1) and

fully

decomposition decreased a minor

amount

of

and

these

two

biopterin

amounts

2.

component.

specifically that

oxidized

processed,

of

The expected

Since

(4) in nearly

1 and

4

in an equal

amount

were

with

(3) was

(1) was

4, it is most

generated

directly

reaction.

A similar,

nation

was

quinonoid

6-methyldihydropterin.

NMR

analysis'

by

of the target

the disproportionation

two

double

doublets

ddd

signal

at 4.52 ppm

appeared

as

adjacent

resonance

at higher

nearby.

These

p-quinonoid 6-methyl

at 3.64 ppm

low

location

dt

at

imino

to

phosphate

buffer7 effect

again

4

and

diminished

was

1.9 min

in

in

Can _-.z 332;

(d) Kaufman,

main

(b) Archer, 50,

with

a 0.1 M

a

at

is

chain

4.01

ppm

to

its

due

protons

on

C(7)

nitrogen

atom

product

to be

isomer

same

reaction

typical

decomposition

pathway

a half

Tris

as the

NADPH,

0.9 min

decomposition The

which

non

conditions,

for of

2

in a 0.1 the

products

isomerization

was

enzymatically

and

3 remained

intact.

and Notes 35, 245;

of Tokyo C. W.;

(b) Nagatsu,

Press,

Tokyo,

Benkovic,

(cl Kaufman,

T. "Biochemistry

1973.

S. J. J- Am,

Chem --2 D. J.; Scrimgeour,

M. C.; Vonderschmitt, 1174;

life

buffer, 7 indicating

Other

quantities.

the same

1971,

DeBrosse,

1972,

(31,

the

excess

1. Under

References

104, 6871; --J. Biochem.

and a

side

dd

two

the ortho

anion. 2c,2f

small

by a large

afforded

S. Adv. ---__ Enzymol ___A of Catecholamines", University

1982,

The

a

C(6)

of the oxidation

than

of 2 proceeded

though

R. A.;

for C(71Heq, on the

and on

'H

(2) exhibited

there is no imino

that

(2) rather

1. (a) Kaufman,

2. (al Lazarus,

atom.

7,8_dihydrobiopterin

1,

stereospecifically

(C(2')H) proton

the structure

of the phosphate

and

the

protons

the

analogues.2ar2gr3

The decomposition

acceleration

ppm

of

suggesting

confirmed

6,7-dimethyl

and at 4.03 ppm

3.89

Thus

the complication

dihydrobiopterin

tertiary

nitrogen

dihydropterins,2b12cf2d12f

at pH 7.0.

virtually

the

results

The two

dihydrobiodisproportio-

3

from

as

stereo-

to consider

slower,

suffered

Quinonoid

resonance

fileds,

Isomerization

were

like

field to

inevitably

for C(7)Hax

for C(6)H.

(GE)-dihydrobiopterin

and

quinonoid

compound products.

a quintet

The

(C(l')Hl.

M

with

but

to the

detected

quinonoid

by a disproportionation observed

As the

formed

rational

from

pterin

also

amounts.

proportionally

7,8-dihydrobiopterin

(6Rl-tetrahydrobiopterin

compounds

equal

increased

S. ------L J. Biol.

Chem

1964,

Sot __L K. G. 239 --I

S. ---~____= J. Biol Chem 1961, 236, 804; (e) Lazarus, R. A.; DeBrosse, C. W.; Benkovic, S. J. -_: J. Am Chem 1982, 104, 6869; (f) sot --~_~ Archer, M. C.; Scrimgeour, K. G. Can J Biochem 1970, 48, 278; (g) -2 2 --___I Benkovic, Am. Chem. 3. Matsuura,

S. J.; Sammons, Sot. 1985,

107,

S; Sugimoto,

4. (a) Matsuura,

S.;

D.; Armarego,

P.; Inners,

R. L J

3706.

T; Murata,

Murata,

W. L. F; Waring,

S;

S. Tetrahedron

Sugimoto,

Lett. 1985,

T. -_-Z Chem

3,

Lett __-_?_ 1984,

4003. 735;

(b)

588

Matsuura,

S.;

Sugimoto,

T.;

Biochem 1985, 98 in press: ----2 Heterocycles in press. 5.

mp

190

'C (dec).

25.40.

Found:

sample

were

6. HPLC

C,

was

detector,

7.26 ml; 7. The

values

were

500 MHz

Chemical

spectrum

individual

'H NMR

in D20

of

2

eluant, flow

Iwasaki,

39.21;

H,

decompositions

of

SCX

phosphate

4.5 x 250 buffer,

Retention

4, 4.36 ml;

volumes

3, 5.02 ml;

based

was

on

the decrease

measured

containing

of the absorbance

alive.

assigned

on a JEOL

University.

GX-500

The

of 1,

at 370

The by the

After 'H

NMR

spectrometer

sample

(CH3)3SiCD2CD2COOD

at 18 "C for 7 min.

1, 3, and 4, was shown

(3.95 mg)

(0.1%)

the observation spectrum,

comparison

with

in the

24 September

19851

and

at was the

completed, which spectra

below.

m N

in Japan

pH

'C.

0

(Received

N, the

(see text).

Partisil-10

2 ml/min.

5.12;

purification

ammonium

H. -.z J T.

S.; Sugimoto,

8.92 ml.

(1.0 ml)

were

column,

2, 3.62 ml;

of Nagoya

still

C,

Further

30 mM rate

Center

was

signals

at18

spectrum

was observed

25%

authentic

follows:

Y.;

S.; Murata,

26.53.

as follows:

calculated

solution

Instrument

dissolved

about

as

NaHS03;

were

Sugawara,

CgH13N503.HC1:

N,

(GS)-tetrahydrobiopterin,

nm in 0.1 mM 8. The

3 mM

S.;

due to the spontaneous

performed

concerned

for

5.06;

UV at 265 nm;

3.2, containing pterins

Calcd H,

unsuccessful

analysis

mm:

Anal. 39.08;

Murata,

(c) Matsuura,

0

m

all of